Method for treating rheumatoid arthritis

ABSTRACT

The present invention provides a method for determining whether a Rheumatoid Arthritis (RA) patient is susceptible to treatment with a B cell targeted therapy, which method comprises the step of analyzing B cells and/or germinal center-like structures (GC-LS) in a synovial tissue sample from the patient; wherein a patient whose synovial tissue sample is B cell rich and/or GC-LS negative is determined to be susceptible to treatment with the B cell targeted therapy, whereas a patient whose synovial tissue sample is B-cell poor and/or GC-LS positive is determined to be resistant to treatment with the B cell targeted therapy.

FIELD OF THE INVENTION

The invention relates to a method for determining whether a rheumatoidarthritis (RA) patient is susceptible to treatment with a B celltargeted therapy, such as Rituximab. The invention also relates to amethod for treating a RA patient who is refractory to B cell targetedtherapy.

BACKGROUND TO THE INVENTION

Inflammatory arthritis is a prominent clinical manifestation in diverseautoimmune disorders including rheumatoid arthritis (RA), psoriaticarthritis (PsA), systemic lupus erythematosus (SLE), Sjogren's syndromeand polymyositis.

RA is a chronic inflammatory disease that affects approximately 0.5 to1% of the adult population in northern Europe and North America. It is asystemic inflammatory disease characterized by chronic inflammation inthe synovial membrane of affected joints, which ultimately leads to lossof daily function due to chronic pain and fatigue. The majority ofpatients also experience progressive deterioration of cartilage and bonein the affected joints, which may eventually lead to permanentdisability. The long-term prognosis of RA is poor, with approximately50% of patients experiencing significant functional disability within 10years from the time of diagnosis. Life expectancy is reduced by anaverage of 3-10 years.

Inflammatory bone diseases, such as RA, are accompanied by bone lossaround affected joints due to increased osteoclastic resorption. Thisprocess is mediated largely by increased local production ofpro-inflammatory cytokines, of which tumor necrosis factor-α (TNF-α) isa major effector.

In RA specifically, an immune response is thought to beinitiated/perpetuated by one or several antigens presenting in thesynovial compartment, producing an influx of acute inflammatory cellsand lymphocytes into the joint. Successive waves of inflammation lead tothe formation of an invasive and erosive tissue called pannus. Thiscontains proliferating fibroblast-like synoviocytes and macrophages thatproduce proinflammatory cytokines such as TNF-α and interleukin-1(IL-1). Local release of proteolytic enzymes, various inflammatorymediators, and osteoclast activation contributes to much of the tissuedamage. There is loss of articular cartilage and the formation of bonyerosions. Surrounding tendons and bursa may become affected by theinflammatory process. Ultimately, the integrity of the joint structureis compromised, producing disability.

B cells are thought to contribute to the immunopathogenesis of RA,predominantly by serving as the precursors of autoantibody-producingcells but also as antigen presenting cells (APC) and pro-inflammatorycytokine producing cells. A number of autoantibody specificities havebeen identified including antibodies to Type II collagen andproteoglycans, as well as rheumatoid factors and most importantly anticitrullinated protein antibodies (ACPA). The generation of largequantities of antibody leads to immune complex formation and theactivation of the complement cascade. This in turn amplifies the immuneresponse and may culminate in local cell lysis.

Current standard therapies for RA which are used to modify the diseaseprocess and to delay joint destruction are known as disease modifyinganti-rheumatic drugs (DMARDs). Methotrexate, leflunomide andsulfasalazine are traditional DMARDs and are often effective asfirst-line treatment.

Biologic agents designed to target specific components of the immunesystem that play role in RA are also used as therapeutics. There arevarious groups of biologic treatments for RA including; TNF-α inhibitors(etanercept, infliximab and adalimumab), human IL-1 receptor antagonist(anakinra) and selective co-stimulation modulators (abatacept).

Rituximab is indicated for the treatment of moderate to severe RA inadult patients who have had an inadequate response to, or cannottolerate, one or more TNF-α inhibitor therapies. It has been shown to beeffective in the treatment of RA in patients refractory to treatmentwith anti-TNF therapy.

The Rituximab antibody is a genetically engineered chimeric murine/humanmonoclonal antibody directed against the CD20 antigen. Rituximab bindshuman complement and lyses lymphoid B-cell lines throughcomplement-dependent cytotoxicity. Additionally, it has significantactivity in assays for antibody-dependent cellular cyotoxicity. Morerecently, Rituximab has been shown to have anti-proliferative effects intritiated thymidine-incorporation assays and to induce apoptosisdirectly. Other anti-CD19 and anti-CD20 antibodies have not been shownto have this activity.

Rituximab treatment has been shown to result in B cell depletion inperipheral blood, bone marrow and the synovium. However, not allpatients refractory to treatment with anti-TNF therapy are responsive toRituximab treatment. Current evidence on the efficacy of Rituximabrelates primarily to rheumatoid factor, ACPA positive patients, althougheven within this population clinical responses are heterogeneous withonly 60% achieving an ACR20 response within 6 months.

Rituximab is associated with various safety issues, especiallyinfusion-related adverse events and is also very expensive, costingapproximately $10,000 per treatment course.

There is thus a need for a method to predict whether a given RA patientis likely to respond to Rituximab treatment. There is also a need foralternative method of treatment for RA patients who are refractory totreatment with Rituximab.

DESCRIPTION OF THE FIGURES

FIG. 1—immunohistochemical staining of tissue biopsy samples used in anexemplar B cell scoring system. Staining for CD20 was used toquantitatively assess the presence of B cells in RA synovial samples,being classified as B cell poor (score from 0 to 1) and B cell rich(score from 2-3) as is shown in the example images. CD21 expression wasassessed in samples displaying a CD20 score of 4 and were positive forGC-LS/FDC networks when a cluster of CD21+ cells was detected. Originalmagnification ×10.

FIG. 2—Atlas 1: Scoring of aggregates on H&E specimens using a 0-3 scale

FIG. 3—Atlas 2: Scoring of CD20, CD138 and CD21 by immunohistochemistryscoring analysis

SUMMARY OF ASPECTS OF THE INVENTION

The present inventors have made the surprising finding that thehistomorphological type of a synovial sample from a RA patient ispredictive of the patient's response to B cell targeted therapy.

Due to the systemic nature of RA, it was thought an assessment ofbiomarkers in peripheral blood was important for identification andstratification of patients who would respond to B cell targeted therapy(Owczarczyk et al. Sci Transl Med 2011; 3:101ra92, Sellam et al.Arthritis & Rheumatism 2011; 63(4):933-938). The present invention,however, now shows that rather than looking at markers in blood, B celllevels and morphology in the synovium are predictive of a RA patient'sresponse to B cell targeted therapy.

In a first aspect, the present invention relates to a method fordetermining whether a Rheumatoid Arthritis (RA) patient is susceptibleto treatment with a B cell targeted therapy, which method comprises thestep of analysing B cells and/or germinal centre-like structures (GC-LS)in a synovial tissue sample from the patient; wherein a patient whosesynovial tissue sample is B cell rich and/or GC-LS negative isdetermined to be susceptible to treatment with the B cell targetedtherapy, whereas a patient whose synovial tissue sample is B-cell poorand/or GC-LS positive is determined to be resistant to treatment withthe B cell targeted therapy.

The step of analysing B cells and/or GC-LS may be performed byhistological analysis. A patient whose synovial tissue sample is B cellpoor may be characterised by a diffuse pattern of a small number of Bcells interspersed with prevalent myeloid inflammatory infiltrate, whilea patient whose synovial tissue sample is B cell rich may becharacterised by the clustering of B cells in an aggregated pattern. Apatient who is GC-LS positive may be characterised by the presence of Bcell aggregates and detection of CD21 within said aggregates.

B cells may be analysed by determining CD20 expression or other markersin the synovial sample.

The B cell targeted therapy may be B cell depletion therapy. The B celltargeted therapy may be selected from the following list: Rituximab,Ocrelizumab, Veltuzumab, Ofatumumab and Epratuzumab.

The B cell targeted therapy may be Rituximab.

A patient whose synovial tissue sample is GC-LS positive may beresistant to Rituximab therapy and determined to be suitable fortreatment with an agent which downregulates IL-6 mediated signalling.The agent that downregulates IL-6 signalling may be an IL-6 receptorantagonist, for example the agent may be Tocilizumab.

The method of the present invention may be performed using a synovialsample from a RA patient who is refractory to synthetic or biologic(e.g. anti-TNF) DMARD therapies.

In a second aspect the present invention provides a kit for use in amethod according to the first aspect.

In a third aspect the present invention provides a method for treating aRheumatoid Arthritis (RA) patient who is refractory to treatment with aB cell targeted therapy which comprises the step of disrupting germinalcentre-like structures (GC-LS) in the synovium.

The GC-LS may be disrupted by treatment with an agent whichdownregulates IL-6 mediated signalling, a proteasome inhibitor or agrowth factor inhibitor.

DETAILED DESCRIPTION

Rheumatoid Arthritis

RA is a chronic, systemic inflammatory disorder that may affect manytissues and organs, but principally attacks synovial joints. It is adisabling and painful condition, which can lead to substantial loss offunctioning and mobility if not adequately treated.

The disease process involves an inflammatory response of the synovium,secondary to massive immune cell infiltration and proliferation ofsynovial cells, excess synovial fluid, and the development of fibroustissue (pannus) in the synovium that attacks the cartilage andsub-chondral bone. This often leads to the destruction of articularcartilage and the formation of bone erosions with secondary ankylosis(fusion) of the joints. RA can also produce diffuse inflammation in thelungs, the pericardium, the pleura, the sclera, and also nodularlesions, most commonly in subcutaneous tissue. RA is considered asystemic autoimmune disease as autoimmunity plays a pivotal role in itschronicity and progression.

The term synovial sample refers to a sample derived from a synovialjoint. Typically the synovial sample will be derived for a synovialjoint of a RA patient. A synovial sample may be a synovial tissue biopsyand the synovial joint may display active inflammation at the time thesample is taken.

A number of cell types are involved in the aetiology of RA, including Tcells, B cells, monocytes, macrophages, dendritic cells and synovialfibroblasts. Autoantibodies known to be associated with RA include thosetargeting Rheumatoid factor (RF) and anti citrullinated proteinantibodies (ACPA).

RA Therapy

A typical patient with newly diagnosed RA is often treated initiallywith nonsteroidal anti-inflammatory drugs and disease-modifyingantirheumatic drugs (DMARDs) such as hydroychloroquine, sulfasalazine,leflunomide, or methotrexate (MTX), alone or in combinations. Patientswho do not respond to general DMARDs may be termed DMARD-refractory.

DMARD-refractory patients are often progressed to biological therapeuticagents, for example TNF-α antagonists such as Adalimumab, Etanercept,Golimumab and Infliximab. Patients who do not respond to TNF-αantagonist therapy may be termed TNF-α antagonist-refractory orinadequate responders (ir).

The method of the present invention may be performed on a synovialsample from a RA patient who has previously been determined to berefractory to DMARD-therapy and/or TNF-α antagonist therapy. The methodmay also be performed on a synovial sample from a RA patient unable totolerate TNF-α antagonist therapy.

B Cells

B cells play a central role in the pathogenesis of RA.

Immature B cells are produced in the bone marrow. After reaching theIgM⁺ immature stage in the bone marrow, these immature B cells migrateto secondary lymphoid tissues (such as the spleen, lymph nodes) wherethey are called transitional B cells, and some of these cellsdifferentiate into mature B lymphocytes and possibly plasma cells.

B cells may be defined by a range of cell surface markers which areexpressed at different stages of B cell development and maturation(Table 1). These B cell markers may include CD19, CD20, CD22, CD23,CD24, CD27, CD38, CD40, CD72, CD79a and CD79b, CD138 and immunoglobulin(Ig).

TABLE 1 Compartment Cell Type Markers Bone marrow Stem cell Pro-B cellCD19⁺ CD20⁻ Ig⁻ Pre-B cell CD19⁺ CD20⁺ Ig⁻ Immature B cell CD19⁺ CD20⁺Ig⁺ Peripheral Naïve B cell CD19⁺ CD20⁺ Ig⁺ CD38^(+/−) compartmentsNaïve activated CD19⁺ CD20⁺ Ig⁺ CD38⁺ B cell GC B cell CD19⁺ CD20⁺ Ig⁺CD38⁺⁺ Post-GC B cell CD19⁺ CD20⁺ Ig⁺ CD38⁺ Memory B cell CD19⁺ CD20⁺Ig^(+/−) CD27⁺ IgM/IgG/IgA⁺ CD38⁻ Plasma blast CD19⁺ CD20⁻ Ig^(+/−)CD27⁺⁺ CD38⁺⁺ Bone marrow Plasma cell CD19^(+/−) CD20⁻ Ig⁻ CD27⁺⁺CD38⁺⁺⁺ CD138⁺

Immunoglobulins (Ig) are glycoproteins belonging to the immunoglobulinsuperfamily which recognise foreign antigens and facilitate the humoralresponse of the immune system. Ig may occur in two physical forms, asoluble form that is secreted from the cell, and a membrane-bound formthat is attached to the surface of a B cell and is referred to as the Bcell receptor (BCR). Mammalian Ig may be grouped into five classes(isotypes) based on which heavy chain they possess. Immature B cells,which have never been exposed to an antigen, are known as naïve B cellsand express only the IgM isotype in a cell surface bound form. B cellsbegin to express both IgM and IgD when they reach maturity—theco-expression of both these immunoglobulin isotypes renders the B cell‘mature’ and ready to respond to antigen. B cell activation followsengagement of the cell bound antibody molecule with an antigen, causingthe cell to divide and differentiate into an antibody producing plasmacell. In this activated form, the B cell starts to produce antibody in asecreted form rather than a membrane-bound form. Some daughter cells ofthe activated B cells undergo isotype switching to change from IgM orIgD to the other antibody isotypes, IgE, IgA or IgG, that have definedroles in the immune system.

CD19 is expressed by essentially all B-lineage cells and regulatesintracellular signal transduction by amplifying Src-family kinaseactivity.

CD20 is a mature B cell-specific molecule that functions as a membraneembedded Ca2⁺ channel. Expression of CD20 is restricted to the B celllineage from the pre-B-cell stage until terminal differentiation intoplasma cells.

CD22 functions as a mammalian lectin for α2,6-linked sialic acid thatregulates follicular B-cell survival and negatively regulates signaling.

CD23 is a low-affinity receptor for IgE expressed on activated B cellsthat influences IgE production.

CD24 is a GPI-anchored glycoprotein which was among the first pan-B-cellmolecules to be identified.

CD27 is a member of the TNF-receptor superfamily. It binds to its ligandCD70, and plays a key role in regulating B-cell activation andimmunoglobulin synthesis. This receptor transduces signals that lead tothe activation of NF-κB and MAPK8/JNK.

CD38 is also known as cyclic ADP ribose hydrolase. It is a glycoproteinthat also functions in cell adhesion, signal transduction and calciumsignalling and is generally a marker of cell activation.

CD40 serves as a critical survival factor for germinal centre (GC) Bcells and is the ligand for CD154 expressed by T cells.

CD72 functions as a negative regulator of signal transduction and as theB-cell ligand for Semaphorin 4D (CD100).

CD79a/CD79b dimer is closely associated with the B-cell antigenreceptor, and enables the cell to respond to the presence of antigens onits surface. The CD79a/CD79b dimer is present on the surface of B-cellsthroughout their life cycle, and is absent on all other healthy cells.

CD138 is also known as Syndecan 1. Syndecans mediate cell binding, cellsignalling, and cytoskeletal organization. CD138 may be useful as a cellsurface marker for plasma cells.

The method of the present invention comprises the step of analysing thepresence of B cells in a synovial sample from a RA patient anddetermining if a RA patient is B cell rich or B cell poor. This analysismay involve determining the presence of cells expressing one or more ofthe markers detailed in Table 1.

The presence of B cells may be determined by analysing the level andpattern of B cells in a synovial sample from a RA patient. Such analysismay be performed by histological analysis.

The identification of RA patients who are B cell rich or B cell poor maybe performed by using a system for grading lymphocytic aggregates knownto those skilled in the art. In the examples section, the presentinventors use a system adapted from the one described in their previouspublication (Manzo et al. Eur J Immunol. (2005); 35(5):1347-1359). Theradial cell count is estimated by counting the number of cells from themore centrally located vessel to the identifiable edge of its aggregate.The determination is made at the point of widest infiltration.

The maximum lymphocytic aggregate identified may be graded according tothe maximum radial cell count measured from the central vessel asdescribed by Manzo et al. (as above). Aggregates may be categorised intothree groups according to the radial cell count as shown in Table 5 andthe grading atlas provided as FIGS. 2 and 3.

TABLE 5 Grade Radial Cell Count 0 No aggregates visualisedUninflamed/Diffuse 1 2-5 cells 2 6-10 cells 3 >10 cells

Depending on the numbers of B cell identified per high power field(HIT), biopsies are thus classified as B cell poor (Grade 0 and 1), Bcell rich (Grade 2 and 3) and Germinal Center (GC) rich Grade 4+CD 21positivity (see Atlas 1, FIG. 2).

Such a grading system may also be used in combination with analysis of Bcell surface markers, for example CD20 and/or CD79a (see Atlas 2, FIG.3).

The presence of B cells within a synovial sample from a RA patient maybe determined by analysing the level of B cell markers within the sampleusing techniques such as next-generation sequencing, gene expressionarrays, PCR and proteomics. Such techniques may be used assess the levelof a B cell marker RNA and/or protein within the synovial sample, withan increased level of the B cell marker determining a B cell richprofile. The B cell marker may be, for example, CD20 and/or CD79a. Thesynovial sample may be a synovial tissue biopsy.

Germinal Centre-Like Structures (GC-LS)

The method of the present invention may comprise the step of analysingthe presence of GC-LS in a synovial sample from a RA patient.

Germinal centres are sites where mature B cells rapidly proliferate,differentiate, and undergo somatic hypermutation and class switchrecombination during an immune response. During this process of rapiddivision and selection, B cells are known as centroblasts, and once theyhave stopped proliferating they are known as centrocytes. B cells withingerminal centres express CD138 when they differentiate into plasmacells. Germinal centres develop dynamically after the activation of Bcells by T-cell dependent antigen.

The term GC-LS refers to an ectopic or tertiary lymphatic structure thatforms in non-lymphoid tissues and may develop to become a place ofautoantibody generation. In the context of RA, the GC-LS form in thesynovium. GC-LS are typically characterised by the presence ofaggregated T and/or B lymphocytes alongside follicular dendritic cells(FDCs).

FDCs have high expression of complement receptors CR1 and CR2 (CD35 andCD21 respectively) and Fc-receptor FcγRIIb (CD32). Further FDC specificmolecular markers include FDC-M1, FDC-M2 and C4.

The identification of GC-LS in a synovial sample of a RA patient maytherefore involve determining the presence of cells positive for one ormore of the above markers. For example it may involve determining thepresence of plasma cells (CD138⁺) and/or FDCs (CD35⁺-CD21⁺).

The identification of GC-LS may be performed using theimmunohistochemistry scoring analysis as provided in Atlas 2 (FIG. 3).

Determining the presence of GC-LS in a synovial sample of a RA patientmay involve identifying FDCs within B cell aggregates using one or moreof the above markers. Determining the presence of GC-LS may involve theidentification of CD21⁺ cells within B cell aggregates in a synovialsample from a RA patient.

B Cell Targeted Therapy

A RA patient identified as B cell rich and/or GC-LS negative by themethod of the present invention is determined as being susceptible totreatment with a B cell targeted therapy whereas a patient who is B-cellpoor and/or GC-LS positive is determined as resistant to treatment witha B cell targeted therapy.

A B cell targeted therapy refers to the administration of an agent thatinterferes with or inhibits the development and/or function of B cells.The B cell targeted therapy may cause B cell depletion or the inhibitionof B cell development and maturation. Advantageously, the B celltargeted therapy is directed against B cells in all stages ofdevelopment other than undifferentiated stem cells and terminallydifferentiated antibody-producing plasma cells.

The agent may be a small molecule drug, such as a Bruton's tyrosinekinase (BTK) inhibitor or other agent which targets B cell signallingpathways.

Direct depletion of B cells may be performed through the use ofmonoclonal antibodies (mAbs) directed against cell surface markers (e.g.CD20 and CD22). Such mAbs bind to the target antigen and kill the cellby initiating a mixture of apoptosis, complement dependent cytotoxicity(CDC), and antibody-dependent cell-mediated cellular cytotoxicity(ADCC).

The B cell targeted therapy used in the present invention may be anagent directed against CD20, for example Rituximab, Ocrelizumab,Veltuzumab or Ofatumumab, or an agent directed against CD22 such asEpratuzumab.

Rituximab is a chimeric mouse/human immunoglobulin G1 (IgG1) monoclonalantibody to CD20 that stimulates B cell destruction upon binding toCD20. Rituximab depletes CD20 surface-positive naïve and memory B cellsfrom the blood, bone marrow and lymph nodes via mechanisms which includeantibody-dependent cellular cytotoxicity (ADCC), complement dependentcytotoxicity (CDC). It does not affect CD20-negative early B celllineage precursor cells and late B lineage plasma cells in the bonemarrow.

Ocrelizumab is a humanized anti-CD20 monoclonal antibody that causesCD20⁺ B cell depletion following binding to CD20 via mechanismsincluding ADCC and CDC.

Veltuzumab is a humanized, second-generation anti-CD20 monoclonalantibody that causes CD20⁺ B cell depletion following binding to CD20via mechanisms including ADCC and CDC.

Ofatumumab is a human monoclonal IgG1 antibody to CD20 and may inhibitearly-stage B lymphocyte activation. Ofatumumab targets a differentepitope located closer to the N-terminus of CD20 compared to the epitopetargeted by rituximab and includes an extracellular loop, as it binds toboth the small and large loops of the CD20 molecule. Ofatumumabstimulates B cell destruction through ADCC and CDC pathways.

Epratuzumab is a humanized monoclonal IgG1 antibody to CD22. It containsa murine sequence comprising 5-10% of the molecule, the remainder beinghuman framework sequences. Epratuzumab binds to the CD22 thirdextracellular domain (epitope B), without blocking the ligand bindingsite, with measured affinity of K_(d)=0.7 nm. In vitro studies showedepratuzumab induces CD22 phosphorylation by binding to its surface. Itresults in modulation, mostly negative, of BCR activation.

IL-6 Mediated Signalling

In a further aspect, a RA patient identified by the method of thepresent invention as GC-LS positive is determined to be suitable fortreatment with an agent which downregulates interleukin-6 (IL-6)signalling.

IL-6 is a cytokine that provokes a broad range of cellular andphysiological responses, including inflammation, hematopoiesis, andoncogenesis by regulating cell growth, gene activation, proliferation,survival, and differentiation. It is able to directly influence B cellactivation state and late stage differentiation towards plasma cells.

IL-6 signals through a receptor composed of two different subunits, analpha subunit that produces ligand specificity and GP (Glycoprotein)130, a receptor subunit shared in common with other cytokines in theIL-6 family. Binding of IL-6 to its receptor initiates cellular eventsincluding activation of JAK (Janus Kinase) kinases and activation ofRas-mediated signalling. Activated JAK kinases phosphorylate andactivate STAT transcription factors, particularly STAT3 and SHP2.Phosphorylated STAT3 then forms a dimer and translocates into thenucleus to activate transcription of genes containing STAT3 responseelements. STAT3 is essential for GP130-mediated cell survival and G1 toS cell-cycle-transition signals. Both c-Myc and Pim have been identifiedas target genes of STAT3 and together can compensate for STAT3 in cellsurvival and cell-cycle transition. SHP2 links cytokine receptor to theRas/MAP (Mitogen-Activated Protein) kinase pathway and is essential formitogenic activity.

The Ras-mediated pathway, acting through SHC, GRB2 (Growth FactorReceptor Bound protein-2) and SOS1 (Son of Sevenless-1) upstream andactivating MAP kinases downstream, activates transcription factors suchas Elk1 and NF-IL-6 (C/EBP-β) that can act through their own cognateresponse elements in the genome.

In addition to JAK/STAT and Ras/MAP kinase pathways, IL-6 also activatesPI3K (Phosphoinositide-3 Kinase). The PI3K/Akt/NF-KappaB cascadeactivated by IL-6, functions cooperatively to achieve the maximalanti-apoptotic effect of IL-6 against TGF-β. The anti-apoptoticmechanism of PI3K/Akt is attributed to phosphorylation of the BCL2family member BAD (BCL2 Associated Death Promoter) by Akt. Thephosphorylated BAD is then associated with 14-3-3, which sequesters BADfrom BCLXL, thereby promoting cell survival. Regulating the BCL2 familymember is also considered as one of the anti-apoptotic mechanisms ofSTAT3, which was may be capable of inducing BCL2 in pro-B cells. Thetermination of the IL-6-type cytokine signaling is through the action oftyrosine phosphatases, proteasome, and JAK kinase inhibitors SOCS(Suppressor of Cytokine Signaling), PIAS (Protein Inhibitors ofActivated STATs), and internalization of the cytokine receptors viaGP130.

An agent which downregulates IL-6 signalling may interfere with orinhibit any of the above stages involved in IL-6 mediated signallingsuch that IL-6 signalling and responses are diminished. For example theagent may be an IL-6 receptor antagonist such as Tocilizumab, which is ahumanized monoclonal antibody against the IL-6 receptor. An IL-6receptor antagonist refers to an agent that reduces the level of IL-6that is able to bind to the IL-6 receptor.

The present invention also provides a method for treating a RA patientwho is refractory to treatment with a B cell targeted therapy, whichcomprises the step of disrupting GC-LS in the synovium.

The GC-LS may be disrupted by treatment with an agent whichdownregulates IL-6 mediated signalling, for example Tocilizumab.

Tocilizumab is a humanized monoclonal IgG1 antibody against the IL-6receptor that binds to soluble and membrane-bound IL-6 receptor.Tocilizumab inhibits the induction of biological activity due to IL-6 incells that have expressed both membrane-bound IL-6 receptor and gp130molecules, and also inhibits the induction of biological activity due toIL-6/IL-6 receptor complex foiuiation in cells that express gp130 alone.Furthermore, since it has the capacity to dissociate IL-6/IL-6 receptorcomplexes that have already formed, it is able to block IL-6 signaltransduction.

The GC-LS may be disrupted by treatment with a growth factor inhibitoror an agent that inhibits signalling required for B cell function. Theagent may, for example, inhibit B-cell activating factor (BAFF) or aproliferation-inducing ligand (APRIL) signalling. Examples of suchagents include, but are not limited to, Belimumab and Atacicept.

Belimumab is a human monoclonal IgG1λ antibody that inhibits BAFF (alsoknown as B-lymphocyte stimulator (BLyS)). BAFF is a 285-amino acid typeII protein that is a member of the TNF ligand superfamily and is a vitalB cell survival factor, with important roles in the differentiation ofimmature to mature B cells and in immunoglobulin class switching andproduction. Belimumab inhibits B cell survival and differentiation byneutralizing soluble BAFF, without directly causing B cell death.

Atacicept is a fully human recombinant fusion protein containing theextracellular ligand-binding portion of the TACI (TransmembraneActivator and CAML [calcium-modulator andcyclophilin-ligand]-Interactor) receptor and a modified Fc portion ofhuman IgG. Atacicept, therefore, contains the binding portion of areceptor that binds both BAFF and APRIL. APRIL is a structural homologueof BAFF that is secreted as a soluble protein by monocytes, macrophages,dendritic cells, neutrophils and T cells, and shares some of thebiological properties of BAFF.

The GC-LS may be disrupted by a proteasome inhibitor such as Bortezomib.

Bortezomib is an N-protected dipeptide that binds the catalytic site ofthe 26S proteasome, thereby inhibiting proteasome function. Theproteasome regulates protein expression and function by degradation ofubiquitylated proteins, and also removes abnormal or misfolded proteinsfrom the cell.

Kit

The present invention also provides a kit for use in a method ofdeteimining whether a RA patient is susceptible to treatment with a Bcell targeted therapy, which method comprises the step of analysing thepresence of B cells and/or germinal centre-like structures (GC-LS) in asynovial tissue sample from the patient;

wherein a patient whose synovial tissue sample is B cell rich and/orGC-LS negative is determined to be susceptible to treatment with the Bcell targeted therapy, whereas a patient whose synovial tissue sample isB-cell poor and/or GC-LS positive is determined to be resistant totreatment with the B cell targeted therapy.

The kit may comprise;

-   -   i) An agent for the detection of B cells in a synovial sample of        a RA patient,    -   ii) An agent for the detection of GC-LS in a synovial sample of        a RA patient.

Agents for the detection of B cells in a synovial sample from a RApatient may include agents that detect CD20 and/or CD79a.

Agents for the detection of GC-LS in a synovial sample of a RA patientmay include agents that detect CD21.

The kit may also comprise instructions for use.

The kit may also comprise a B cell targeted therapy or an agent thatdownregulates IL-6 signalling.

The invention will now be further described by way of Examples, whichare meant to serve to assist one of ordinary skill in the art incarrying out the invention and are not intended in any way to limit thescope of the invention.

EXAMPLES Example 1—An Investigation into the Molecular MechanismsPredicting Response to Rituximab in RA Patients Resistant to Anti-TNFTherapy

Aims

This was an open-label, ultrasound guided synovial biopsy-based study ofRA patients. The primary aim was to investigate in anti-TNFa inadequateresponders whether baseline synovial pathobiological phenotypes definedspecific response/resistance subsets to Rituximab therapy.

Results

Patient demographics of 27 patients recruited are shown in Table 2.

TABLE 2 Patient demographics of total study population (n = 27) No (%)Female 21 (77) Rheumatoid factor +ve 24 (88) CCP +ve 25 (97) Erosive 25(97) Mean +/− St Dev Age 59.1 (+/−14.1) Baseline DAS 6.1 (+/−1.6)

A preliminary analysis of the first 21 patients recruited to the studyclassified patients into either an aggregate or diffuse synovialhistological pattern and numbers of responders/non-responders withineach histological group were determined (Table 2). Results were analysedusing Fisher's exact test and demonstrated a significant difference inresponse between groups (p=0.014), with a significantly higherpercentage of aggregate (83%) versus diffuse (20%) patients respondingto Rituximab treatment.

TABLE 3 Response to Rituximab is significantly associated with anaggregate histological pattern and non-response is significantlyassociated with a diffuse infiltrate in TNF inadequate responders (n =21, p = 0.014). Clinical response Synovial Histomorphological Pattern toRituximab Aggregate number (%) Diffuse number (%) Responder 5 (83)  3(20) Non-responder 1 (17) 12 (80)

All 27 patients were classified as B cell rich, B cell poor or germinalcentre like structures (GC-LS) positive (+ve). Numbers of responders andnon-responders within each histological group were identified andresults analysed using Fisher's exact test (Table 4). A significantdifference (p=0.032) was seen between the percentage of patientsresponding to Rituximab in the B cell poor (22%) versus B cell rich(80%) group. In addition within the group of patients classified asGC-LS positive there was a trend towards non-response to Rituximab (75%non-responders vs 25% responders).

TABLE 4 Immunohistological classification of synovial tissue as B cellrich, B cell poor or Germinal centre demonstrates a significantdifference in levels of response to Rituximab within B cell rich and Bcell poor patients (n = 27, p = 0.032). Synovial HistomorphologicalPattern Clinical response Germinal Centre +ve B cell B cell to Rituximabn (%) rich n (%) poor n (%) Responder 1 (25) 4 (80)  4 (22)Non-responder 3 (75) 1 (20) 14 (78)

Materials and Methods

Patients and Samples

At baseline patients underwent a minimally invasive ultrasound guidedsynovial biopsy of an actively inflamed joint. Baseline clinical datawas collected including age, sero positivity for rheumatoid factor,anti-CCP antibody status, disease activity score (DAS) of 28 joint countand radiographs of hands and feet. Patients were infused with standardRituximab therapy (2× 1 g Rituximab therapy at a fortnightly interval)within 2 weeks of the biopsy. DAS-28 assessment along with routinebloods following Rituximab therapy were performed on a monthly basis for12 months. Patients were classified as responders to treatment at 3months according to EULAR response criteria and responders were eligiblefor re-treatment at 6 and 12 month time points. Non-responders toRituximab were switched to other biologic drugs according to NICEguidelines (TA195 and TA198).

The primary outcome measure was the number of responders at 3 monthswithin each histomorphological subgroup.

Histological Grading of Tissues

Synovial tissue specimens were immediately fixed in 4% formalin. Afterparaffin embedding, 3 μm serial sections underwent routine H&E stainingin order to define the predominant histological pattern of RA synovitisas either diffuse or aggregate. Lymphocytic aggregates were classifiedinto three groups based on a scoring system the present inventors hadadapted from their previous publication (Manzo et al. Eur J Immunol.(2005); 35(5):1347-1359). Depending on the numbers of B cell identifiedper high power field (BPF), biopsies were classified as B cell poor(Grade 0 and 1), B cell rich (Grade 2 and 3) and Geiminal Center (GC)rich Grade 4+CD 21 positive.

In addition, formalin-fixed, paraffin-embedded tissue sections underwentimmunohistochemical staining. Following antigen retrieval with Targetretrieving solution (DAKO) sections were stained for CD20 (IgG2a, cloneL26; DAKO) at an antibody dilution of 1:20 and following proteinase Kdigestion (DAKO) single staining for CD21 (IF8, DAKO) at a dilution of1:20 was also performed to identify FDC networks (CD21 +ve)characterising germinal centre like structures (GC-LS). Based on theresults of the CD21 staining and the presence of G2/G3 aggregates,samples were qualitatively classified as either B cell poor (diffuse),or B cell rich (CD21-ve) or germinal centre positive (CD21 +ve) (FIG.1). The grading was performed at ×10 magnification over the wholesublining area available for each section and by 2 independent observerswith excellent correlation (Intra class correlation coefficient forrepeated measurements of B cell score 0.96).

Statistical Analysis

Numbers of patients classified as responders and non-responders withineach histological group was determined and significant differencesbetween groups analysed using Fisher's exact test. P values <0.05 weretaken as significant. Intra class correlation coefficients were used todetermine reliability of B cell score between two observers.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled incellular biology, histology or related fields are intended to be withinthe scope of the following claims.

The invention claimed is:
 1. A method for treating a RheumatoidArthritis (RA) patient susceptible to treatment with a B cell targetedtherapy, which method comprises the steps of: (a) identifying an RApatient susceptible to treatment with a B cell targeted therapy byanalysing B cells and/or germinal centre-like structures (GC-LS) in asynovial tissue sample from an RA patient, wherein analysing B cells andGC-LS is performed by histological analysis; wherein a patient whosesynovial tissue sample is B cell rich and/or GC-LS negative issusceptible to treatment with the B cell targeted therapy; wherein the Bcell rich synovial tissue sample comprises a B cell aggregate with aradial cell count of 6 cells or greater measured from a central vesselat the point of widest infiltration; wherein the GC-LS negative synovialtissue sample lacks B cell aggregates within which CD21 is detectable;and (b) administering a B cell targeted therapy to the RA patientsusceptible to treatment with the B cell targeted therapy.
 2. The methodaccording to claim 1, wherein B cells are analysed by determining CD20expression in the synovial tissue sample.
 3. The method according toclaim 1, wherein the B cell targeted therapy is B cell depletiontherapy.
 4. The method according to claim 1, wherein the B cell targetedtherapy is selected from the group consisting of: Rituximab,Ocrelizumab, Veltuzumab, Ofatumumab and Epratuzumab.
 5. The methodaccording to claim 4, wherein the B cell targeted therapy is Rituximab.6. The method according to claim 1, wherein the RA patient is refractoryto anti-TNF therapy.